The goal of this study is to elucidate the molecular mechanisms critical to maintain the regulated activity and localization of CHT at presynaptic cholinergic nerve terminals using structural and proteomics based approaches. CHT is maintained on synaptic vesicles and is recruited for activity at the presynaptic membrane in periods of demand for the essential acetylcholine precursor, choline. The molecular basis for this unique protein trafficking is partly based on a basal endocytosis motif. However, observed sustained CHT plasma membrane activity in vivo can not be entirely explained by the rapid basal activity of this motif. This proposal will utilize functional and endocytosis assays of CHT to uncover additional regulatory motifs in CHT cytoplasmic sequence. The basis for these assays is the sensitivity of CHT to the activation of kinase linked pathways, specifically PKC and PKA. Structural analysis will identify sequence motifs that mediate regulatory endocytic control of CHT. The culmination of these finding will be the examination of interactions between the basal and regulated endocytic motifs in modulating CHT trafficking and cell surface activity, and verification of a conserved mechanism of trafficking in cultured neuronal preparations. The proteomics based approach will determine protein binding partners of CHT in the presynaptic terminal that have a role in the regulation of CHT activity at the plasma membrane and its vesicular localization. Striatal preparations will be combined with in-vitro binding techniques to capture associated proteins of CHT. The identity of captured associations will be discerned by LC/MS analysis and verified by established biochemical methodologies. Protein associations that occur at the basal and regulatory endocytosis motifs will be explored by alterations in the in-vitro binding preparations. This analysis of CHT will describe the interaction between structural trafficking motifs and protein binding partners in the regulation of CHT activity and trafficking in the presynaptic terminal. There is great demand for improved cholinergic therapies and this research will provide new targets for therapeutic interventions by characterizing this newly defined molecular target critical for cholinergic neuronal function. Public: It is well established that disruption of cholinergic signaling in vivo induces cognitive deficits in man and augmentation of cholinergic tone enhances attention, learning and memory in aging and dementias. The tone of cholinergic signaling is determined by pre-synaptic choline (Ch) levels limited by CHT (Ch transporter) activity. This research will define mechanisms that regulate CHT activity in cholinergic neurons and will contribute to the public health initiative to improve cholinergic therapies. [unreadable] [unreadable] [unreadable]